Numerous diagnostic, analytical and preparative procedures include steps in which temperature changes are effected. To achieve reproducible and accurate results it is required to maintain control of the temperature of a reaction mixture, such as a sample, and to maintain temperature uniformity within the respective reaction mixture. Further, many diagnostic, analytical and preparative procedures rely on enzymes, which show an optimal performance at a defined temperature. To obtain exact temperature control it is generally required to provide a close contact between the reaction mixture and the heating or cooling element. At the same time cross-contaminations of different reaction mixtures, e.g. samples, need to be avoided.
An example of a process in which controlled heating and maintenance of temperature uniformity are particularly important is in vitro nucleic acid amplification by means of the polymerase chain reaction (PCR). Typically the PCR process is a thermal cycling process, wherein the basic cycle can be divided into three steps: (a) separation of the DNA double-strand at about 90° C. to about 94° C. (b) cooling down to about 50° C.-70° C. to renaturate the specific primers to the single stranded DNA (annealing), and (c) increasing the temperature to about 70° C.-80° C. for extension of the primers with thermostable DNA polymerase (elongation).
Temperature control during a PCR reaction is typically performed by a feedback loop system, while temperature uniformity is achieved by highly thermally conductive but bulky materials such as copper. In addition to temperature control and maintenance of temperature uniformity within the sample, it is also important to provide a sample heating (cooling) rate of at least 5 K/s (−5 K/s). A high heating rate is accomplished by the implementation of a Proportional Integrated Derivative (PID) control system limited by maximum dissipated power and heat capacitance. A high cooling rate is rather difficult to achieve and bulky systems require force cooling by either a Thermoelectric Element (TEC, often called Peltier element) or by other means, such as water. Such devices are complicated and power hungry.
As the systems are bulky, their thermal time constants are in minutes rather than seconds. That results in long transition times and unwanted by-products of the PCR reaction. The high power consumption furthermore eliminates the possibility of making a battery-operated and portable PCR system. In addition, the reaction tubes are large and the required amount of PCR cocktail renders the entire process cost-intensive. Furthermore, the detection of PCR product has to be done off-line, i.e. by employing another device, resulting in additional costs.
While the systems currently used to carry out PCR reactions allow for running multiple samples at the same time, they do not allow for individual temperature control of different samples. Where it is desired to expose samples to varying temperature cycle conditions, several systems therefore have to be employed in parallel. It is therefore desirable to provide an apparatus that is able to simultaneously handle samples individually during PCR.
Miniaturization of devices in the chemical, pharmaceutical and biotechnological field has lead to the development of microfluidic devices and microarrays. Accordingly, micro PCR methods (μPCR) are being developed, which are expected to become a central part of a Lab-on-a-Chip or micro Total Analysis Systems (μTAS). Two basic approaches can be identified, one being a stationary system with cycling temperature, the other being a flow system with three zones at different temperatures.
Stationary systems cycle the temperature of the chamber in order to modify the temperature of the PCR solution. They do not require a pumping system or other means of transfering the PCR sample. The flow-through systems typically have zones at three constant temperatures, between which the sample is moved and thus changes its temperature. While being faster than the stationary system, the flow-through system requires an implementation of a mechanism to transfer between the zones of different temperature. In either case a heater is integrated into the PCR system, so it is not economical to dispose of the device to avoid crosscontamination after performing only a single test.
A recent example of a stationary micro PCR method uses a planar chip device and the formation of a Virtual Reaction Chamber (VRC). The VRC is made by encapsulation of a water based sample in oil (Guttenberg, Z., et al., Lab Chip, 2005, 5, 308-317). As no solid cover or microchannels are required, the device fabrication consists only of deposition and patterning thin film heaters and temperature sensors on a suitable substrate. The respective device is however still too costly for a disposable system.
A further challenge occurring upon miniaturization is the risk of cross-contamination between samples. The safest way of avoiding such cross-contamination is the use of a disposable system. At the very least, the part of the device which comes into contact with the sample should be disposable. So far, many different systems have been proposed. These systems typically do not fulfil all the requirements listed above and they are relatively expensive. An approach with a disposable part made of a plastic sheet has been disclosed in U.S. Pat. No. 6,509,186. A set of wells is formed by hot embossing and the whole set is placed on top of heaters. This system employs a relatively complicated microfabrication process and the disposable plate needs to be customized. Therefore, there remains a need for a μPCR that is simple to manufacture, easy to operate, and economical enough to be disposable. The optional ability to be integrated into a complete μTAS system is highly desirable.
Accordingly it is an object of the present invention to provide an apparatus and a method for regulating the temperature of a chemical and/or biological sample which avoids these discussed disadvantages.